The present invention relates to the cloning and characterization of a human serine/threonine kinase (h-sgk: serum and glucocorticoid dependent kinase). The invention furthermore relates to reagents for diagnosing conditions associated with a change in cell volume and/or in "macromolecular crowding" in the body, such as, for example, hypernatremia, hyponatremia, diabetes mellitus, renal failure, hypercatabolism, hepatic encephalopathy, inflammation and microbial or viral infections. The present invention additionally relates to pharmaceuticals comprising the h-sgk, nucleic acids which code for the h-sgk, or receptors, in particular antibodies, which specifically bind to the h-sgk.
Even when the extracellular osmolarity is constant, the constancy of the cell volume is continuously challenged due to transport across cell membranes and cellular metabolism, i.e. production and breakdown of osmotically active substances.
Cell swelling and shrinkage disturb the intracellular environment by diluting and concentrating, respectively, cellular macromolecules which lead to extensive impairment of cellular functions. This is why cells have developed a large number of cell volume-regulating mechanisms. Cell swelling leads, in most tissues, to cellular release of ions due to activation of ion channels and KCl cotransport. Cell shrinkage conversely leads to cellular uptake of ions due to activation of NaCl/KCl cotransporter and Na.sup.+ /H.sup.+ exchanger.
Furthermore, cell shrinkage stimulates cellular accumulation and cell swelling stimulates cellular release of osmolytes, molecules which are specifically used to generate intracellular osmolarity [Burg, M. B., Am. J. Physiol. 268: F983-F996, 1995].
Finally, changes in the liver cell volume influence hepatocellular metabolism and gene expression [Haussinger et al. (1994) Am. J. Physiol. 267, E343-E355]. Cell swelling acts like an anabolic signal which stimulates protein and glycogen synthesis and inhibits protein and glycogen breakdown. Conversely, cell shrinkage acts as a catabolic signal by promoting the breakdown of glycogen and proteins and inhibiting the synthesis of proteins and glycogen [Haussinger et al. (1994) Am. J. Physiol. 267, E343-E355].
The cell volume has been recognized as a crucial element in the regulation of hepatocellular metabolism by hormones, cellular amino acid uptake and oxidative stress.
The signal mechanisms which couple cell function to the changes in the cellular hydration state are substantially unknown. Changes in the cell volume achieve their various effects on cell function partly by stimulating or suppressing the expression of particular genes, whose products then influence the expression or activity of a large number of cell components. In order to discover genes which are increasingly expressed on cell swelling, we carried out a differential mRNA fingerprinting assay on cDNAs from hepatocytes which had been exposed either to isotonic or to anisotonic extracellular fluid. This resulted in a plurality of bands which showed differential expression rates on use of different primers.
It has been found, surprisingly, that the expression of one of these bands was stimulated under hypertonic conditions and inhibited under hypotonic conditions. The cDNA sequence of this band, whose expression is influenced in a particular way by changes in cell volume, has been analyzed in detail. It was found by sequence comparison that there is no similarity with any previously known human gene. The gene which has been found, whose nucleotide sequence is depicted in FIG. 1, surprisingly codes for a kinase, a putative serine/threonine kinase. Its sequence is depicted in FIG. 2 as well as in FIG. 1. It is highly homologous with previously known rat sgk (serum and glucocorticoid dependent kinase), a kinase whose expression is increased by serum and glucocorticoids. A dependence of the rat sgk on cell volume has not previously been described, however.
The present invention accordingly relates to a human cell volume-regulated kinase (h-sgk) and to processes for producing it by genetic manipulation.
Expression of the h-sgk is greatly dependent on the cell volume. Cell swelling inhibits expression of the h-sgk, whereas cell shrinkage stimulates expression of the h-sgk. Furthermore, expression of the h-sgk is inhibited by urea. Urea impairs, like changes in cell volume, the stability and thus the function of cellular proteins and the packing density of the cellular macromolecules, called macromolecular crowding [Minton, A. P., Mol. Cell. Biochem. 55: 119-140, 1983]. h-sgk expression is therefore a measure of the cellular macromolecular crowding. Transcription of the h-sgk is not, in contrast to rat sgk, influenced either by corticoids or by fetal calf serum (FCS), however.
The h-sgk is expressed in a large number of human tissues such as liver, heart, pancreas, muscle, kidney, lung, placenta, lymphocytes and several structures in the brain (hippocampus, nucleus caudatus, corpus callosum, substantia nigra, nucleus subthalamicus and thalamus).
It has emerged that the h-sgk has a considerable diagnostic potential for many diseases in which changes in cell volume play a crucial pathophysiological part. Expression of the h-sgk can be demonstrated by detecting and/or quantifying the mRNA by using suitable probes, for example in a Northern blot or by in situ hybridization, and the h-sgk itself can be detected, for example, using suitable antibodies in a Western blot or by immunohistochemistry. Suitable probes and antibodies have already been successfully checked for utilizability.
The present invention therefore also relates to the diagnostic use of the h-sgk, its fragments or the relevant nucleic acids coding therefor. The diagnostic techniques which can be used are known to the skilled worker. These may be all immunoassay formats known from the prior art, such as, for example, Western blot or enzyme linked immunosorbent assay (ELISA), but also homogeneous assay formats not bound to a solid phase. Conceivable examples are competitive assay variants, but also indirect assays or designs on the sandwich principle are also directly possible. It is likewise possible to employ the labeling techniques known to the skilled worker. All types of nucleic acid detection techniques can be used, such as, for example, Southern blot, Northern blot and all variants of the hybridization techniques, including in situ hybridization.
The h-sgk can be detected both in body fluids, for example, blood, plasma or serum, and in solid tissues, for example biopsy material. Detection of the h-sgk is indicated wherever changes in the cell volume or in the macromolecular crowding in the body occur, such as in hypernatremia, hyponatremia, diabetes mellitus, renal failure, hypercatabolism, hepatic encephalopathy, inflammation and infections.
Furthermore, dysfunction of the h-sgk might lead to impaired regulation of hepatic metabolism. Detection of the h-sgk would therefore be useful for diagnostic elucidation of fructose intolerance and hyper- and hypoglycemic states.
Hypernatremia: This is a life-threatening disturbance which occurs, for example, when there is osmotic diuresis and water diuresis due to central or nephrogenic diabetes insipidus. Central diabetes insipidus results from a genetic defect, craniocerebral trauma, damage to hypothalamic neurons due to inflammations, hypoperfusion, tumors, consumption of alcohol, opiates and some drugs. Nephrogenic diabetes insipidus results from genetic defects, hypokalemia, hypercalcemia, protein deficiency, pyelonephritis, and treatment with various drugs etc. As is shown in experiments on cultivated liver and kidney cells, an increase in the extracellular Na.sup.+ concentration, which is always associated with an increase in the extracellular osmolarity too, results in increased expression of the h-sgk. The kinase can thus be used as indicator of the extent of cell shrinkage and be employed for monitoring the therapy. Surveillance of this type is important inasmuch as fatal cell swelling may occur on occasion if the correction of hypernatremia is too rapid, despite extracellular hyperosmolarity.
Hyponatremia: Hyponatremia below 130 mmol/l is found in about 1-2% of all hospitalized patients. The causes of this life-threatening disturbance are diabetes mellitus, ketonuria, hepatic insufficiency, diuretics, opiates, various drugs, osmotic diuresis, bicarbonaturia, adrenal insufficiency, salt-loss nephritis, nephrotic syndrome, increased secretion of ADH and losses of isotonic fluid (for example diarrhea) with replacement only of water. If the hyponatremia is the result of an increase in other osmolytes in the blood, then the cell volume and expression of the h-sgk remain normal. However, if the hyponatremia reflects a hypoosmolarity with cell swelling, then there is a reduction in h-sgk expression.
Thus, measurement of the h-sgk provides information about the presence of cell swelling and allows a rational decision to be made about the therapeutic procedure. The kinase can be employed to check progress during therapy. Correction of hyponatremia which is too rapid may result in cell shrinkage, which is occasionally fatal.
Diabetes mellitus: Hyperglycemia occurs in diabetes mellitus and results in an increase in the extracellular osmolarity and thus causes cell shrinkage. The glucose undergoing glomerular filtration exceeds the maximum renal transport rate and, in this way, forces osmotic diuresis, in which Na.sup.+ and water are lost. This may result in development of hyponatremia. The increased extracellular osmolarity and the oversupply of glucose promote the cellular production of sorbitol which, when the extracellular osmolarity falls, results in cell swelling. The cell shrinkage and cell swelling associated with diabetes mellitus are ascribed crucial importance in the pathophysiology [McManus et al., New England J. Med. 333: 1260-1266, Dermadash et al., Kidney intern. 50: 2032-2040, 1996]. Measurement of the h-sgk in a patient with diabetes mellitus permits the changes in cell volume to be estimated and thus provides a solid basis for compensating electrolyte disturbances. In this case too, observation of the progress can prevent excessive corrections.
Renal failure: In renal failure there is a massive increase in the urea concentration to levels which have a destabilizing effect on proteins, cause cells to shrink and bring about a decrease in h-sgk expression. The destabilizing effect of urea is diminished by the formation of trimethylamines. When the changes in the urea concentration are rapid, the accumulation of trimethylamines cannot keep up, and disturbances of cellular metabolism are to be expected owing to the changes in cell volume. Determination of the h-sgk may reveal an imbalance between destabilizing urea and stabilizing trimethylamines. Therapeutic administration of trimethylamines would, where appropriate, be indicated if the h-sgk is greatly depressed.
Hypercatabolism: In a number of catabolic states, such as sepsis, burns, acute pancreatitis, major operations, changes in the volume of muscle cells correlating with the extent of hypercatabolism have been detected. Cell shrinkage in fact leads to enhancement, and cell swelling to inhibition, of proteolysis. Determination of the h-sgk might justify in the individual case the use of therapeutic measures suitable for counteracting cell shrinkage, such as administration of glutamine [Haussinger et al., Lancet, 341: 1330-1332, 1993] or of osmolytes [Burg, M. B., J. Exp. Zool. 268(2): 171-5, 1994].
Hepatic encephalopathy: There is compelling evidence that hepatic encephalopathy is brought about by swelling of glial cells [Norenberg, M. D., Exp. Neurol. 53(3): 213-220, 1994]. It is in fact possible to detect a decrease in the osmolyte inositol in the brain in cases of liver disease [Kreis et al., NMR Biomed. 4: 109-116, 1991]. Complete disappearance coincides with the onset of encephalopathy. Development and use of suitable substrates for the h-sgk might allow the h-sgk activity in the brain to be measured and counter-regulated even before the encephalopathy occurs. Where appropriate, h-sgk expression in more readily accessible tissues might also be used as indicator of volume changes in glial cells.
Alzheimer disease: Recent evidence points to increase of peripheral cell volume in Alzheimer disease. Moreover, the osmolyte inositol is enhanced in patients with Alzheimer disease, but not in dementia of other causes. H-sgk expression may contribute to diagnosis of Alzheimer disease.
Infections/Inflammation: Sepsis is associated with extensive cell shrinkage [Haussinger et al., Lancet 1993, 341: 1330-1332] with the corresponding occurrence of hypercatabolism. In fact, the cell volume plays an important part in the pathogen-host relationship. Expression of the h-sgk might be a valuable parameter for assessing the pathophysiology of infections. In situ hybridisation reveals marked increase of tissue levels of h-sgk in inflammatory diseases, such as hepatitis, pancreatitis, Morbus Crohn, or glomerulonephritis. Moreover, h-sgk expression is enhanced by TGF.beta. which has been implicated in progressive fibrosis such as liver cirrhosis, lung fibrosis and progressive renal failure. H-sgk expression has indeed been found enhanced in patients with chronic renal failure.
Hyperglycemia/hypoglycemia/lactacidosis: Diminished or enhanced expression and/or function of the h-sgk might result in disturbances of carbohydrate metabolism as observed in association with cell shrinkage and cell swelling [Lang et al., Pflugers Arch. 413: 209-216, 1989]. A diminished function would result in the threat of hypoglycemia. Enhanced function might be followed, on the one hand, by hyperglycemia or, on the other hand, by lactacidosis. Thus, in the diagnostic elucidation of hyperglycemia, hypoglycemia and lactacidoses of unclear origin, it would always also be expedient to investigate the expression and function of the h-sgk.
The present invention is additionally explained further by the following detailed description and, furthermore, described by the examples and the claims.